Assessing the Response of Small RNA Populations to Allopolyploidy Using Resynthesized Brassica napus Allotetraploids.

Allopolyploidy, combining interspecific hybridization with whole genome duplication, has had significant impact on plant evolution. Its evolutionary success is related to the rapid and profound genome reorganizations that allow neoallopolyploids to form and adapt. Nevertheless, how neoallopolyploid genomes adapt to regulate their expression remains poorly understood. The hypothesis of a major role for small noncoding RNAs (sRNAs) in mediating the transcriptional response of neoallopolyploid genomes has progressively emerged. Generally, 21-nt sRNAs mediate posttranscriptional gene silencing by mRNA cleavage, whereas 24-nt sRNAs repress transcription (transcriptional gene silencing) through epigenetic modifications. Here, we characterize the global response of sRNAs to allopolyploidy in Brassica, using three independently resynthesized Brassica napus allotetraploids originating from crosses between diploid Brassica oleracea and Brassica rapa accessions, surveyed at two different generations in comparison with their diploid progenitors. Our results suggest an immediate but transient response of specific sRNA populations to allopolyploidy. These sRNA populations mainly target noncoding components of the genome but also target the transcriptional regulation of genes involved in response to stresses and in metabolism; this suggests a broad role in adapting to allopolyploidy. We finally identify the early accumulation of both 21- and 24-nt sRNAs involved in regulating the same targets, supporting a posttranscriptional gene silencing to transcriptional gene silencing shift at the first stages of the neoallopolyploid formation. We propose that reorganization of sRNA production is an early response to allopolyploidy in order to control the transcriptional reactivation of various noncoding elements and stress-related genes, thus ensuring genome stability during the first steps of neoallopolyploid formation.

Meiotic gene evolution: can you teach a new dog new tricks?

Meiosis, the basis of sex, evolved through iterative gene duplications. To understand whether subsequent duplications have further enriched the core meiotic "tool-kit," we investigated the fate of meiotic gene duplicates following whole genome duplication (WGD), a common occurrence in eukaryotes. We show that meiotic genes return to a single copy more rapidly than genome-wide average in angiosperms, one of the lineages in which WGD is most vividly exemplified. The rate at which duplicates are lost decreases through time, a tendency that is also observed genome-wide and may thus prove to be a general trend post-WGD. The sharpest decline is observed for the subset of genes mediating meiotic recombination; however, we found no evidence that the presence of these duplicates is counterselected in two recent polyploid crops selected for fertility. We therefore propose that their loss is passive, highlighting how quickly WGDs are resolved in the absence of selective duplicate retention.

Molecular evolution and transcriptional regulation of the oilseed rape proline dehydrogenase genes suggest distinct roles of proline catabolism during development.

MAIN CONCLUSION: Six BnaProDH1 and two BnaProDH2 genes were identified in Brassica napus genome. The BnaProDH1 genes are mainly expressed in pollen and roots' organs while BnaProDH2 gene expression is associated with leaf vascular tissues at senescence. Proline dehydrogenase (ProDH) catalyzes the first step in the catabolism of proline. The ProDH gene family in oilseed rape (Brassica napus) was characterized and compared to other Brassicaceae ProDH sequences to establish the phylogenetic relationships between genes. Six BnaProDH1 genes and two BnaProDH2 genes were identified in the B. napus genome. Expression of the three paralogous pairs of BnaProDH1 genes and the two homoeologous BnaProDH2 genes was measured by real-time quantitative RT-PCR in plants at vegetative and reproductive stages. The BnaProDH2 genes are specifically expressed in vasculature in an age-dependent manner, while BnaProDH1 genes are strongly expressed in pollen grains and roots. Compared to the abundant expression of BnaProDH1, the overall expression of BnaProDH2 is low except in roots and senescent leaves. The BnaProDH1 paralogs showed different levels of expression with BnaA&C.ProDH1.a the most strongly expressed and BnaA&C.ProDH1.c the least. The promoters of two BnaProDH1 and two BnaProDH2 genes were fused with uidA reporter gene (GUS) to characterize organ and tissue expression profiles in transformed B. napus plants. The transformants with promoters from different genes showed contrasting patterns of GUS activity, which corresponded to the spatial expression of their respective transcripts. ProDHs probably have non-redundant functions in different organs and at different phenological stages. In terms of molecular evolution, all BnaProDH sequences appear to have undergone strong purifying selection and some copies are becoming subfunctionalized. This detailed description of oilseed rape ProDH genes provides new elements to investigate the function of proline metabolism in plant development.

Homoeologous duplicated regions are involved in quantitative resistance of Brassica napus to stem canker.

BACKGROUND: Several major crop species are current or ancient polyploids. To better describe the genetic factors controlling traits of agronomic interest (QTL), it is necessary to understand the structural and functional organisation of these QTL regions in relation to genome duplication. We investigated quantitative resistance to the fungal disease stem canker in Brassica napus, a highly duplicated amphidiploid species, to assess the proportion of resistance QTL located at duplicated positions. RESULTS: Genome-wide association analysis on a panel of 116 oilseed rape varieties genotyped with 3228 SNP indicated that 321 markers, corresponding to 64 genomic regions, are associated with resistance to stem canker. These genomic regions are relatively equally distributed on the A (53%) and C (47%) genomes of B. napus. Overall, 44% of these regions (28/64) are duplicated homoeologous regions. They are located in duplications of six (E, J, R, T, U and W) of the 24 ancestral blocks that constitute the B. napus genome. Overall, these six ancestral blocks have 34 duplicated copies in the B.napus genome. Almost all of the duplicated copies (82% of the 34 regions) harboured resistance associated markers for stem canker resistance, which suggests structural and functional conservation of genetic factors involved in this trait in B. napus. CONCLUSIONS: Our study provides information on the involvement of duplicated loci in the control of stem canker resistance in B. napus. Further investigation of the similarity/divergence in sequence and gene content of these duplicated regions will provide insight into the conservation and allelic diversity of the underlying genes.

High-density SNP-based genetic map development and linkage disequilibrium assessment in Brassica napus L.

BACKGROUND: High density genetic maps built with SNP markers that are polymorphic in various genetic backgrounds are very useful for studying the genetics of agronomical traits as well as genome organization and evolution. Simultaneous dense SNP genotyping of segregating populations and variety collections was applied to oilseed rape (Brassica napus L.) to obtain a high density genetic map for this species and to study the linkage disequilibrium pattern. RESULTS: We developed an integrated genetic map for oilseed rape by high throughput SNP genotyping of four segregating doubled haploid populations. A very high level of collinearity was observed between the four individual maps and a large number of markers (>59%) was common to more than two maps. The precise integrated map comprises 5764 SNP and 1603 PCR markers. With a total genetic length of 2250 cM, the integrated map contains a density of 3.27 markers (2.56 SNP) per cM. Genotyping of these mapped SNP markers in oilseed rape collections allowed polymorphism level and linkage disequilibrium (LD) to be studied across the different collections (winter vs spring, different seed quality types) and along the linkage groups. Overall, polymorphism level was higher and LD decayed faster in spring than in "00" winter oilseed rape types but this was shown to vary greatly along the linkage groups. CONCLUSIONS: Our study provides a valuable resource for further genetic studies using linkage or association mapping, for marker assisted breeding and for Brassica napus sequence assembly and genome organization analyses.

Complementarity and discriminatory power of genotype and otolith shape in describing the fine-scale population structure of an exploited fish, the common sole of the Eastern English Channel.

Marine organisms show population structure at a relatively fine spatial scale, even in open habitats. The tools commonly used to assess subtle patterns of connectivity have diverse levels of resolution and can complement each other to inform on population structure. We assessed and compared the discriminatory power of genetic markers and otolith shape to reveal the population structure on evolutionary and ecological time scales of the common sole (Solea solea), living in the Eastern English Channel (EEC) stock off France and the UK. First, we genotyped fish with Single Nucleotide Polymorphisms to assess population structure at an evolutionary scale. Then, we tested for spatial segregation of the subunits using otolith shape as an integrative tracer of life history. Finally, a supervised machine learning framework was applied to genotypes and otolith phenotypes to probabilistically assign adults to subunits and assess the discriminatory power of each approach. Low but significant genetic differentiation was found among subunits. Moreover, otolith shape appeared to vary spatially, suggesting spatial population structure at fine spatial scale. However, results of the supervised discriminant analyses failed to discriminate among subunits, especially for otolith shape. We suggest that the degree of population segregation may not be strong enough to allow for robust fish assignments. Finally, this study revealed a weak yet existing metapopulation structure of common sole at the fine spatial scale of the EEC based on genotypes and otolith shape, with one subunit being more isolated. Our study argues for the use of complementary tracers to investigate marine population structure.

Comparative genomic analysis of duplicated homoeologous regions involved in the resistance of Brassica napus to stem canker.

All crop species are current or ancient polyploids. Following whole genome duplication, structural and functional modifications result in differential gene content or regulation in the duplicated regions, which can play a fundamental role in the diversification of genes underlying complex traits. We have investigated this issue in Brassica napus, a species with a highly duplicated genome, with the aim of studying the structural and functional organization of duplicated regions involved in quantitative resistance to stem canker, a disease caused by the fungal pathogen Leptosphaeria maculans. Genome-wide association analysis on two oilseed rape panels confirmed that duplicated regions of ancestral blocks E, J, R, U, and W were involved in resistance to stem canker. The structural analysis of the duplicated genomic regions showed a higher gene density on the A genome than on the C genome and a better collinearity between homoeologous regions than paralogous regions, as overall in the whole B. napus genome. The three ancestral sub-genomes were involved in the resistance to stem canker and the fractionation profile of the duplicated regions corresponded to what was expected from results on the B. napus progenitors. About 60% of the genes identified in these duplicated regions were single-copy genes while less than 5% were retained in all the duplicated copies of a given ancestral block. Genes retained in several copies were mainly involved in response to stress, signaling, or transcription regulation. Genes with resistance-associated markers were mainly retained in more than two copies. These results suggested that some genes underlying quantitative resistance to stem canker might be duplicated genes. Genes with a hydrolase activity that were retained in one copy or R-like genes might also account for resistance in some regions. Further analyses need to be conducted to indicate to what extent duplicated genes contribute to the expression of the resistance phenotype.

Plant genetics. Early allopolyploid evolution in the post-Neolithic Brassica napus oilseed genome.

Oilseed rape (Brassica napus L.) was formed ~7500 years ago by hybridization between B. rapa and B. oleracea, followed by chromosome doubling, a process known as allopolyploidy. Together with more ancient polyploidizations, this conferred an aggregate 72× genome multiplication since the origin of angiosperms and high gene content. We examined the B. napus genome and the consequences of its recent duplication. The constituent An and Cn subgenomes are engaged in subtle structural, functional, and epigenetic cross-talk, with abundant homeologous exchanges. Incipient gene loss and expression divergence have begun. Selection in B. napus oilseed types has accelerated the loss of glucosinolate genes, while preserving expansion of oil biosynthesis genes. These processes provide insights into allopolyploid evolution and its relationship with crop domestication and improvement.

The genome of the mesopolyploid crop species Brassica rapa.

We report the annotation and analysis of the draft genome sequence of Brassica rapa accession Chiifu-401-42, a Chinese cabbage. We modeled 41,174 protein coding genes in the B. rapa genome, which has undergone genome triplication. We used Arabidopsis thaliana as an outgroup for investigating the consequences of genome triplication, such as structural and functional evolution. The extent of gene loss (fractionation) among triplicated genome segments varies, with one of the three copies consistently retaining a disproportionately large fraction of the genes expected to have been present in its ancestor. Variation in the number of members of gene families present in the genome may contribute to the remarkable morphological plasticity of Brassica species. The B. rapa genome sequence provides an important resource for studying the evolution of polyploid genomes and underpins the genetic improvement of Brassica oil and vegetable crops.